Methods and compounds for treating diseases caused by reactive oxygen species

ABSTRACT

Provided is a method of treating a patient having an inflammatory disease by using a compound which inhibits the complex I-mediated ROS production, a medicament containing such compound and methods for screening for such compounds.

CROSS REFERENCE TO RELATED APPLICATION

This application is a 35 U.S.C. 371 National Phase Entry Applicationfrom PCT/EP2007/007754, filed Sep. 5, 2007.

The present invention refers to a method for treating diseases, inparticular immune diseases dependent on the CD95/CD95L signal system, byinhibiting the generation of reactive oxygen species (ROS).

INTRODUCTION

Under physiological conditions, reactive oxygen species (ROS) form as anatural byproduct of the normal metabolism of oxygen and have importantroles in cell signaling. They are generally very small molecules and arehighly reactive due to the presence of unpaired valence shell electrons.ROS include oxygen ions, free radicals and peroxides both inorganic andorganic. During times of environmental stress, ROS levels can increasedramatically, which can result in significant damage to cell structures,resulting in a pathological condition known as oxidative stress. Cellsare normally able to defend themselves against ROS damage through theuse of enzymes such as superoxide dismutases (SOD) and catalases. Smallmolecule antioxidants such as ascorbic acid (vitamin C), uric acid, andglutathione also play important roles as cellular antioxidants.Similarly, polyphenol antioxidants assist in preventing ROS damage byscavenging free radicals. The effects of ROS on cell metabolism includenot only roles in programmed cell death and apoptosis, but also positiveeffects such as the induction of host defense genes and mobilization ofion transport systems. In particular, platelets involved in wound repairand blood homeostasis release ROS to recruit additional platelets tosites of injury, providing a link to the adaptive immune system via therecruitment of leukocytes.

ROS are implicated in cellular activity to a variety of inflammatoryresponses including cardiovascular diseases. They may also be involvedin hearing impairment via cochlear damage induced by elevated soundlevels, ototoxicity of drugs such as cisplatin, and in congenitaldeafness in both animals and humans. Redox signaling is also implicatedin mediation of apoptosis or programmed cell death and ischemic injury.Specific examples include stroke and heart attack.

Even though to date a clear connection can be drawn from an excess ROSproduction to certain immunological disorders resulting fromintracellular signaling processes, the molecular source and thesignalling steps necessary for ROS production are largely unknown. It isknown in the art that ROS play a key role in regulation of ActivationInduced T cell Death (AICD) by induction of CD95L expression. SinceCD95L expression is crucial for induction of AICD, efforts have beenmade to explore the connection between T-cell receptor (TCR) signallingand regulation of CD95L transcription. Following TCR engagement thekinase ZAP70 is activated (11). ZAP70 phosphorylates the adaptor proteinLAT (19) which recruits phospholipase C gamma 1 (PLCγ1) subsequently.The activation of PLCγ1 results in generation of inositol3,4,5-triphosphate (IP₃) and diacylglycerol (DAG). IP₃ mediates anincrease of cytosolic calcium (Ca²⁺), whereas DAG activates PKC. Therise in cytosolic Ca²⁺ causes activation of the transcription factornuclear factor of activated T cells (NF-AT) (69), one of the keyregulators of CD95L expression (41). In addition, ROS are shown to becrucial for activation-induced CD95L expression (7, 15, 25) possibly viathe ROS inducible transcription factors NF-κB and AP-1 (17). Aerobicorganisms produce ROS by several means; in mitochondria as by-product ofrespiration (63), at the endoplasmatic reticulum by cytochrome P450(50), in the cytoplasm by xanthine oxidase (20), at the plasma membraneby NADPH oxidases (35, 46) and phospholipases (54), and in peroxisomes(56). Recently, the phagocytic NADPH oxidase (NOX2) has been shown to beone source for TCR-triggered ROS. However, NOX2 is not the only sourcefor activation-induced ROS (30). Following T cell activation,respiratory activity increases (21) and mitochondrial ROS production maybe enhanced (27). In addition, there are hints supporting a possiblerole of the mitochondrial electron transport chain (ETC) and cytochromeP450 as origins of activation-induced ROS (7).

Among others, WO2004017959 discloses treatment methods usingaryl-substituted heterocyles which are capable of preventing ROSformation by inhibiting the so-called Fenton reaction, i.e. the reactionbetween hydroxide peroxide and reduced iron, by inhibiting the reductionof iron(III) to iron(II). Furthermore, WO9836748 describes the use ofL-ergothioneine in preventing mitochondria from oxidative stress causedby enhanced ROS. However, none of the documents of the prior artprovides methods and compounds that exert an inhibitory effect withouthazarding the consequences of deleterious side effects when ROSproduction is, in principle, may be desired.

Consequently, there is a need for treatment methods and compounds whichact precisely on the site of deleterious ROS production that gives riseto AICD via the CD95/CD95L signalling system, leading to a variety ofdiseases associated with the cells of the immune system, and, todirectly and specifically inhibit the production of ROS.

The inventors of the instant invention have now surprisingly found thatmetformin, a compound which prevents mitochondrial complex I—mediatedROS production, can be used to inhibit the expression of the CD95 ligandand, as a consequence, also inhibits the phenomenon of AICD. Similarresults were obtained when siRNA-mediated knockdown of the chaperoneNDUFAF1, which is required for complex I assembly, was used forinhibition of complex I—mediated reverse electron flux. Thus, metforminor other inhibitors of complex I—mediated ROS production can be used asa pharmaceutical to treat diseases which are a result of an increasedCD95/CD95 L signalling.

DETAILED DESCRIPTION

The problem of the prior art is solved by the present invention, whichprovides a method of treating a patient having a disease dependent on adysfunction of the CD95/CD95L signal system, and a method of treating apatent having an inflammatory disease, the method comprisingadministering to said patient a therapeutically effective amount of acompound which inhibits complex I-mediated ROS production.

For the purpose of convenience, the term “compound which inhibitscomplex I-mediated ROS production” is hereinafter referred to as“complex I inhibitor”.

As used herein, “reactive oxygen species” is synonym for oxygen radicalsand hydrogen peroxide (H₂O₂), pro-oxidants and refers to molecules orions formed by the incomplete one-electron reduction of oxygen. Thesereactive oxygen intermediates include singlet oxygen; superoxides;peroxides; hydroxyl radical; and hypochlorous acid. They contribute tothe microbicidal activity of phagocytes, regulation of signaltransduction and gene expression, and the oxidative damage to nucleicacids; proteins; and lipids. Preferably, the ROS the production of whichshall be inhibited is hydrogen peroxide.

As used herein, “complex I” refers to an enzyme complex, also known asNADH—ubiquinone oxidoreductase (EC 1.6.5.3). The enzyme couples thetransfer of two electrons from NADH to ubiquinone to the translocationof four protons across the mitochondrial inner membrane. The thusgenerated proton gradient is used by complex V to produce ATP. Mammaliancomplex I consists of 46 polypeptide subunits, seven encoded by themitochondrial DNA and the remainder by the nuclear genome, anon-covalently bound flavomononucleotide (FMN) group and eight ironsulphur.

As used herein, “inhibiting complex I” means inhibiting, decreasing orabolishing the complex-I mediated process of electron transfer. The term“electron transfer” is synonym for “electron flux”. Encompassed hereinis the forward electron flux as well as the reverse electron flux.Because it can be envisaged that inhibiting the forward electron fluxmay be accompanied by undesired side effects, the use of a compound thatinhibits the reverse electron flux is generally preferred.

An assay of how to measure reverse electron flux and the successfulinhibition thereof, respectively, is described in Batandier et al.(Journal of Bioenergetics and Biomembranes, 2006, vol. 38, Nr. 1, pp.33-42), which is incorporated herein by reference. Other examplesinclude Hinkle et al., Journal of Biological Chemistry, 1967, Vol. 242,page 5169; or Grivennikova and Vinogradov, Biochimica et Biophysica Acta1757 (2006) 553-561.

General complex I inhibitors that may be employed for the method of thepresent invention include, e.g., rotenone, piericidin A, ubicidin-3,rollinisatatin-1 and 2 (bullatacin), capsaicin, annonaceous acetogenins,pyridaben, fenpyroximate, fenazaquin, tebufenpyrad, substitutedquinolones and quinolines, synthetically simplified deguelin compounds,antimycin A (AntA), myxothiazol (Myx), and hybrid structures ofcomplex-I and complex-III inhibitors (“chromone derivatives”);acetogenines such as annonacin, and biguanides, such as phenformin,buformin and metformin.

Most preferably, the complex I inhibitor that may be employed for themethod of the present invention is metformin.

The metformin which can be employed to enable the teaching of thepresent invention is well-described in the art. As used herein, the term“metformin” means metformin base or any pharmaceutically acceptable salte.g., metformin hydrochloride and dibasic salts such as metforminfumarate and metformin succinate, originally described in U.S. Pat. No.3,174,901. Commonly used is metformin as different salts thereof, mostlyas hydrochloride salt. Also included is an acetylsalicyl acid salt orclofibrate salt as described in U.S. Pat. No. 3,957,853 and U.S. Pat.No. 4,080,472, which are incorporated herein by reference. Metformin isapproved and commonly known as a pharmaceutical to treatnon-insulin-dependent diabetes mellitus (NIDDM, type II diabetes). It isavailable under its trade name Glucophage® by Bristol Myers Squibb (seefor example for reference US2007141154). Also encompassed for the methodof the present invention are formulations described in US2007160671,which is incorporated by reference herein as well.

In another embodiment of the present invention, the inhibition ofcomplex I can be achieved by destabilizing complex I as a whole, i.e.the complex of 46 subunits, or by destabilizing single subunits thereof.Preferably, the destabilization method affects the complex as a whole.It is known in the art that large protein complexes, like complex I, arekept in its conformational folding state by the aid of so-calledchaperones. Chaperones are proteins that assist the non-covalentfolding/unfolding and the assembly/disassembly of macromolecularstructures, but do not occur in these structures when the latter areperforming their normal biological functions. Human mitochondrialcomplex I assembly is mediated by NDUFAF1 (NADH dehydrogenase(ubiquinone) 1 alpha subcomplex, assembly factor 1) (see reference no.66). Thus, complex I destabilization is preferably achieved by usingsiRNA for the purpose of interfering with/down-regulating the expressionof NDUFAF1, leaving other, rather pan-acting chaperones unaffected.

As used herein, the term “treating” or “treatment” as used in relationto the treatment of diseases, particularly inflammatory diseases is tobe understood as embracing both symptomatic and prophylactic modes, thatis the immediate treatment, e.g. of acute inflammation (symptomatictreatment) as well as advance treatment to prevent, ameliorate orrestrict long term symptomatology (prophylactic treatment). The term“treatment” as used in the present specification and claims in relationto such diseases is to be interpreted accordingly as including bothsymptomatic and prophylactic treatment, e.g., in the case of asthma,symptomatic treatment to ameliorate acute inflammatory events andprophylactic treatment to inhibit ongoing inflammatory status and toameliorate future bronchial exacerbation associated therewith.

As used herein, the term “inflammatory disease” refers to diseases whichare a result of an individual's reaction of connecting tissue and bloodvessels to an external or internal inflammation stimulus, with thepurpose to eliminate or inactivate said stimulus. Inflammationtriggering effectors may include mechanical stimuli, or other physicalfactors, e.g. ionizing radiation, UV light, heat, coldness; chemicalsubstances, e.g. bases, acids, heavy metals, bacterial toxins,allergens, and immune complexes, as well as pathogens, e.g.microorganisms and viruses, worms and insects; and pathogenic metabolismproducts, malfunctioning enzymes, malign tumors.

In a particular embodiment, the term “inflammatory disease” refers to adisease that results from a activation-dependent expression of CD95and/or CD95 ligand (CD95L). In a further embodiment, the term“inflammatory disease” refers to a disease resulting from an increasedexpression of cytokines such as IL-2, IL-4, or TNF-alpha. It has beenshown by the inventors of the present invention that the expression ofeither CD95L or the cytokines described above can be reduced byinhibiting complex I-mediated ROS production with metformin or othercomplex I inhibitors.

As used herein, “therapeutically effective amount” of the complex Iinhibitor means a sufficient amount of said compound to treat aparticular disease, at a reasonable benefit/risk ratio. In general, theterm “therapeutically effective amount” shall refer to an amount of saidcompound which is physiologically significant and improves anindividual's health. An agent, i.e. said compound, is physiologicallysignificant if its presence results in a change in the physiology of therecipient human. For example, in the treatment of a pathologicalcondition, administration of said compound which relieves or arrestsfurther progress of the condition would be considered bothphysiologically significant and therapeutically effective. Said compoundmay be employed in pure form or, where such forms exist, in“pharmaceutically acceptable salt”, ester or prodrug forms.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgement,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977), which is incorporated hereinby reference. The salts can be prepared in situ during the finalisolation and purification of the complex I inhibitors, or separately byreacting the free base function with a suitable organic acid. Examplesof pharmaceutically acceptable, nontoxic acid addition salts are saltsof an amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, berate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydmxy-ethanesulfonate, lactobionate, lactate,laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate. 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, alkyl sulfonate and aryl sulfonate.

As described supra, the complex I inhibitors are useful in the treatmentof inflammatory diseases, particularly inflammatory diseases which mayarise due to an increased ROS production in response to an external orinternal inflammation stimulus, which in turn leads to an increasedexpression of CD95L as well as cytokine such as 11-2, IL 4, TNF-alpha.Such inflammatory diseases can be subdivided into chronic inflammatorydiseases, acute inflammatory diseases, allergic inflammatory disorders,graft-versus-host rejection diseases, and autoimmune disorders, all ofwhich are encompassed within the present invention. Among thesesubgroups, inflammatory diseases of the respiratory tract, inflammatoryskin diseases, allergic inflammatory disorders, inflammatory diseases ofthe gastrointestinal tract and inflammatory heart diseases can occur.

Accordingly, complex I inhibitors are useful for the treatment ofdiseases or conditions responsive to or requiring anti-inflammatory,immunosuppressive or related therapy, including topical administrationfor the treatment of such diseases or conditions of the eye, nasalpassages, buccal cavity, skin, heart, colon or, especially, airways orlung. In particular complex I inhibitors permit topicalanti-inflammatory, immunosuppressive or related therapy with theconcomitant avoidance or reduction of undesirable systemic side effects,for example renal toxicity or general systemic immunosuppression.

Complex I inhibitors are particularly useful for the treatment ofdiseases and conditions of the airways or lung, i.e diseases of therespiratory tract, in particular inflammatory or obstructive airwaysdisease. They are especially useful for the treatment of diseases orconditions of the airways or lung associated with or characterized byinflammatory cell infiltration or other inflammatory event accompaniedby the accumulation of inflammatory cells, e.g. eosinophils and/orneutrophils.

In this respect, Complex I inhibitors are useful in the treatment ofasthma of whatever type of genesis including both intrinsic and,especially, extrinsic asthma. They are useful for the treatment ofatopic and non-atopic asthma, including allergic asthma, bronchiticasthma, exercise-induced asthma, occupational asthma, asthma inducedfollowing bacterial infection and other non-allergic asthmas. Complex Iinhibitors are also useful for the treatment of bronchitis or for thetreatment of chronic or acute airways obstruction associated therewith.Complex I inhibitors may be used for the treatment of bronchitis ofwhatever type or genesis, including, for example, acute bronchitis,arachidic bronchitis, catarrhal bronchitis, chronic bronchitis, croupousbronchitis, phthinoid bronchitis and so forth. Complex I inhibitors arein addition useful for the treatment of pneumoconiosis (an inflammatory,commonly occupational, disease of the lungs, frequently accompanied byairways obstruction, whether chronic or acute, and occasioned byrepeated inhalation of dusts) of whatever type or genesis, including,for example, aluminosis, anthracosis, asbestosis, berylliosis,chalicosis, ptilosis, siderosis, silicosis, tabacosis and, inparticular, byssinosis. Complex I inhibitors may also be used to treatany disease or condition of the airways or lung requiringimmunosuppressive therapy, e.g., for the treatment of autoimmunediseases of, or as they affect, the lungs (for example, for thetreatment of sarcoidosis, alveolitis or chronic hypersensitivitypneumonitis) or for the maintainance of allogenic lung transplant, e.g.,following lung or heart lung transplantation.

For the above purposes, some complex I inhibitors preferably will beadministered topically within the airways, e.g. by the pulmonary route,by inhalation. While having potent efficacy when administered topically,complex I inhibitors are devoid of, or exhibit relatively reduced,systemic activity, e.g. following oral administration. Complex Iinhibitors thus provide a means for the treatment of diseases andconditions of the airways or lung with the avoidance of unwantedsystemic side effect, e.g., consequent to inadvertent swallowing of drugsubstance during inhalation therapy. (It is estimated that during thecourse of maneuvers required to effect administration by inhalation, upto 90% or more of total drug substance administered will inadvertentlybe swallowed rather than inhaled). By the provision of complex Iinhibitors which are topically active, e.g. effective when inhaled butsystemically inactive, the present invention makes complex I inhibitortherapy available to subjects for whom such therapy might otherwise beexcluded, e.g., due to the risk of systemic, in particularimmunosuppressive, side effects.

Complex I inhibitors are also useful for the treatment of other diseasesor conditions, in particular diseases or conditions having an autoimmuneor inflammatory component and for which topical therapy may bepracticed, for example, treatment of diseases and conditions of the eyesuch as conjunctivitis, keratoconjunctivitis sicca, and vernalconjunctivitis and maintenance of corneal transplant, diseases affectingthe nose including allergic rhinitis, diseases and conditions of theskin including psoriasis, atopic dermatitis, pemphigus and contactdermatitis, as well as diseases of the colon, for example Crohn'sdisease and ulcerative colitis.

As immunosuppressants, complex I inhibitors are useful when administeredfor the prevention of immune-mediated tissue or organ graft rejection.Examples of transplanted tissues and organs which suffer from theseeffects are heart, kidney, liver, medulla ossium, skin, cornea, lung,pancreas, intestinum tenue, limb, muscle, nervus, duodenum, small-bowel,pancreatic-islet-cell, and the like; as well as graft-versus-hostdiseases brought about by medulla ossium transplantation.

The regulation of the immune response by complex I inhibitors would alsofind utility in the treatment of autoimmune disorders, such asrheumatoid arthritis, systemic lupus erythematosis, hyperimmunoglobulinE, Hashimoto's thyroiditis, multiple sclerosis, progressive systemicsclerosis, myasthenia gravis, type I diabetes, uveitis, allergicencephalomyelitis, glomerulonephritis, and the like; and furtherinfectious diseases caused by pathogenic microorganisms, such as HIV. Inthe particular cases of HIV-1, HIV-2 and related retroviral strains,inhibition of T-cell mitosis would suppress the replication of thevirus, since the virus relies upon the host T-cell's proliferativefunctions to replicate.

Further uses include the treatment and prophylaxis of inflammatory andhyperproliferative skin diseases and cutaneous manifestations ofimmunologically-mediated illnesses, such as psoriasis, atopicaldermatitis, contact dermatitis and further eczematous dermatitises,seborrhoeis dermatitis, Lichen planus, Pemphigus, bullous pemphigoid,Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas,cutaneous eosinophilias, Lupus erythematosus, acne and Alopecia areata;various eye diseases (autoimmune and otherwise) such askeratoconjunctivitis, vernal conjunctivitis, keratitis, herpetickeratitis, conical cornea, dystrophia epithelialis comeae, cornealleukoma, ocular pemphigus, Mooren's ulcer, Scleritis, Graves'opthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, multiplemyeloma, etc.; inflammation of mucosa and blood vessels such as gastriculcers, vascular damage caused by ischemic diseases and thrombosis.Moreover, hyperproliferative vascular diseases such as intimal smoothmuscle cell hyperplasia, restenosis and vascular occlusion, particularlyfollowing biologically- or mechanically-mediated vascular injury can betreated or prevented by complex I inhibitors.

Other treatable conditions would include but are not limited toParkinson's disease, ischemic bowel diseases, inflammatory boweldiseases, necrotizing enterocolitis, intestinal lesions associated withthermal burns; intestinal inflammations/allergies such as Coeliacdiseases, proctitis, eosinophilic gastroenteritis, and mastocytosis;food-related allergic diseases which have symptomatic manifestationremote from the gastrointestinal tract (e.g., migraine, rhinitis andeczema); renal diseases such as interstitial nephritis, Goodpasture'ssyndrome, hemolytic-uremic syndrome and diabetic nephropathy; nervousdiseases such as multiple myositis, Guillain-Barre syndrome, Meniere'sdisease, polyneuritis, multiple neuritis, mononeuritis andradiculopathy; endocrine diseases such as hyperthyroidism and Basedow'sdisease; hematic diseases such as pure red cell aplasia, aplasticanemia, hypoplastic anemia, idiopathic thrombocytopenic purpura,autoimmune hemolytic anemia, agranulocytosis, pernicious anemia,megaloblastic anemia and anerythroplasia; bone diseases such asosteoporosis; respiratory diseases such as sarcoidosis, fibroid lung andidiopathic interstitial pneumonia; skin disease such as dermatomyositis,leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity andcutaneous T cell lymphoma; circulatory diseases such asarteriosclerosis, atherosclerosis, aortitis syndrome, polyarteritisnodosa and myocardosis; collagen diseases such as scleroderma, Wegener'sgranuloma and Sjogren's syndrome; adiposis; eosinophilic fasciitis;periodontal disease such as lesions of gingiva, periodontium, alveolarbone and substantia ossea dentis; nephrotic syndrome such asglomerulonephritis; male pattern aleopecia or alopecia senilis bypreventing epilation or providing hair germination and/or promoting hairgeneration and hair growth; muscular dystrophy; Pyoderma and Sezary'ssyndrome; Addison's disease; active oxygen-mediated diseases, as forexample organ injury such as ischemia-reperfusion injury of organs (suchas heart, liver, kidney and digestive tract) which occurs uponpreservation, transplantation or ischemic disease (for example,thrombosis and cardiac infarction): intestinal diseases such asendotoxin-shock, pseudomembranous colitis and colitis caused by drug orradiation; renal diseases such as ischemic acute renal insufficiency andchronic renal insufficiency; pulmonary diseases such as toxinosis causedby lung-oxygen or drug (for example, paracort and bleomycins), lungcancer and pulmonary emphysema; ocular diseases such as cataracta,siderosis, retinitis, pigmentosa, senile macular degeneration, vitrealscarring and corneal alkali burn; dermatitis such as erythemamultiforme, linear IgA ballous dermatitis and cement dermatitis; andothers such as gingivitis, periodontitis, sepsis, pancreatitis, diseasescaused by environmental pollution (for example, air pollution), aging,carcinogenis, metastasis of carcinoma and hypobaropathy; disease causedby histamine release; Behcet's disease such as intestinal-, vasculo- orneuro-Behcet's disease, and also Behcet's which affects the oral cavity,skin, eye, vulva, articulation, epididymis, lung, kidney and so on.

Furthermore, the complex I inhibitors may be used for the treatment ofneurological disorders and injuries, particularly central nervous systeminjuries e.g. brain injuries and/or spinal cord injuries as described inWO2004/71528 which is herein incorporated by reference.

Aqueous liquid compositions of the present invention may be particularlyuseful for the treatment and prevention of various diseases of the eyesuch as autoimmune diseases (including, for example, conical cornea,keratitis, dysophia epithelialis corneae, leukoma, Mooren's ulcer,sclevitis and Graves' ophthalmopathy) and rejection of cornealtransplantation. In particular, compositions pertaining to the presentinvention are useful for treating a subject for immune-mediated organ ortissue allograft rejection, a graft-versus-host disease, an autoimmunedisease, an obstructive airway disease, a hyperproliferative disease, oran ischemic or inflammatory intestinal or bowel disease.

Accordingly, the present invention further refers to pharmaceuticalpreparations for the treatment of inflammatory diseases which comprisesa complex I inhibitor and, optionally, a pharmaceutically acceptablecarrier, and methods for preparing such pharmaceutical compositions. Themethods for preparing pharmaceutical compositions, i.e. medicaments, areknown per se to the skilled artisan.

The specific therapeutically-effective dose level for any particularpatient will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; activity of the specificcomplex I inhibitor employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific complex I inhibitor employed; the duration of the treatment;drugs used in combination or coincidental with the specific complex Iinhibitor employed; and like factors well known in the medical arts. Forexample, it is well within the skill of the art to start doses of thecomplex I inhibitor at levels lower than required to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved.

As used herein, the term “pharmaceutically acceptable carrier” means anon-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Someexamples of materials which can serve as pharmaceutically acceptablecarriers are sugars such as lactose, glucose and sucrose; starches suchas corn starch and potato starch; cellulose and its derivatives such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients such as cocoabutter and suppository waxes; oils such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such a propylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, phosphate buffer solutions; non-toxic,compatible lubricants such as sodium lauryl sulfate and magnesiumstearate; as well as coloring agents, releasing agents, coating agents,sweetening, flavoring and perfuming agents. Preservatives andantioxidants can also be present in the composition, according to thejudgment of the formulator.

The compositions may be administered to humans and other animals orally,rectally, parenterally, intracisternally, intravaginally,intraperitoneally, topically (as by powders, ointments, drops ortransdermal patch), bucally, or as an oral or nasal spray. The term“parenteral” as used herein refers to modes of administration whichinclude intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion.

Dosage forms for topical or transdermal administration of complex Iinhibitors include ointments, pastes, creams, lotions, gels, plasters,cataplasms, powders, solutions, sprays, inhalants or patches. The activecomponent, i.e. the complex I inhibitor, is admixed under sterileconditions with a pharmaceutically acceptable carrier and any neededpreservatives or buffers as may be required. The ointments, pastes,creams and gels may contain, in addition to an active complex Iinhibitor of this invention, excipients such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof. Powders and sprays can contain, in additionto complex I inhibitors, excipients such as lactose, talc, silicic acid,aluminum hydroxide, calcium silicates and polyamide powder, or mixturesof these substances. Sprays can additionally contain customarypropellants such as chlorofluorohydrocarbons. For nasal administration,complex I inhibitors will suitably be administered in liquid or powderedform from a nasal applicator. Forms suitable for ophthalmic use willinclude lotions, tinctures, gels, ointment and ophthalmic inserts, againas known in the art. For rectal administration, i.e., for topicaltherapy of the colon, complex I inhibitors may be administered insuppository or enema form, in particular in solution, e.g., in vegetableoil or like oily system for use as a retention enema.

It is clear that safety may be maximized by delivering the drugs by theinhaled route either in nebuliser form or as dry powder. Clearly thegreat advantage of the inhaled route, over the systemic route, in thetreatment of asthma and other diseases of airflow obstruction and/or ofchronic sinusititis, is that patients are exposed to very smallquantities of the drug and the complex I inhibitor is delivered directlyto the site of action.

Complex I inhibitors therefore are preferably employed in any dosageform appropriate for topical administration to the desired site. Thus,for the treatment of diseases of the airways or lungs, complex Iinhibitors may be administered via the pulmonary route/by inhalationfrom an appropriate dispenser device. For this purpose, complex Iinhibitors may be employed in any suitable finely dispersed or finelydispersible form, capable of administration into the airways or lungs,for example in finely divided dry particulate form or in dispersion orsolution in any appropriate (i.e., pulmonarily administerable) solid orliquid carrier medium. For administration in dry particulate form,complex I inhibitors may, for example, be employed as such, i.e., inmicronised form without any additive materials, in dilution with otherappropriate finely divided inert solid carrier or diluent (e.g.,glucose, lactose, mannitol, sorbitol, ribose, mannose or xylose), incoated particulate form or in any other appropriate form as known in theart for the pulmonary administration of finely divided solids. Pulmonaryadministration may be effected using any appropriate system as known inthe art for delivering drug substance in dry or liquid form byinhalation, e.g. an atomizer, nebulizer, dry-powder inhaler or likedevice. Preferably a metered delivery device, i.e., capable ofdelivering a pre-determined amount of complex I inhibitor at eachactuation, will be employed. Such devices are known in the art.

Pharmaceutical compositions of this invention for parenteral injectioncomprise pharmaceutically-acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, as well as sterilepowders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), carboxymethylcellulose and suitable mixturesthereof, vegetable oils (such as olive oil), and injectable organicesters such as ethyl oleate. Proper fluidity may be maintained, forexample, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

These compositions may also contain adjuvants such as preservative,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents such as sugars, sodium chloride,and the like. Prolonged absorption of the injectable pharmaceutical formmay be brought about by the inclusion of agents which delay absorption,such as aluminium monostearate and gelatine.

In some cases, in order to prolong the effect of the drug, it isdesirable to slow the absorption of the drug from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle.

Injectable depot forms are made by forming mieroencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide,poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drugto polymer and the nature of the particular polymer employed, the rateof drug release can be controlled. Depot injectable formulations arealso prepared by entrapping the drug in liposomes or microemulsionswhich are compatible with body tissues. The injectable formulations maybe sterilized, for example, by filtration through a bacterial-retainingfilter, or by incorporating sterilizing agents in the form of sterilesolid compositions which can be dissolved or dispersed in sterile wateror other sterile injectable medium just prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecomplex I inhibitor is mixed with at least one inert,pharmaceutically-acceptable excipient or carrier, such as sodium citrateor dicalcium phosphate and/or a) fillers or extenders such as starches,lactose, sucrose, glucose, mannitol, and silicic acid, b) binders suchas, for example, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose, and acacia, c) humectants such asglycerol, d) disintegrating agents such as agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and sodiumcarbonate, e) solution retarding agents such as paraffin, f) absorptionaccelerators such as quaternary ammonium compounds, g) wetting agentssuch as, for example, cetyl alcohol and glycerol monostearate, h)absorbents such as kaolin and bentonite clay, and i) lubricants such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof. In the case of capsules,tablets and pills, the dosage form may also comprise buffering agents.Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules may be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart, They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Liquid dosage forms for oral administration includepharmaceutically-acceptable emulsions, solutions, suspensions, syrupsand elixirs. In addition to the active complex I inhibitors, the liquiddosage forms may contain inert diluents commonly used in the art suchas, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethyl formamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Suspensions may contain,in addition to the active complex I inhibitors, suspending agents as,for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitoland sorbitan esters, microcrystalline cellulose, aluminum metahydroxide,bentonite, agar-agar, and tragacanth, and mixtures thereof.

Topical administration includes administration to the skin or mucosa,including surfaces of the lung and eye. Compositions for topicaladministration, including those for inhalation, may be prepared as a drypowder which may be pressurized or non-pressurized. In non-pressurizedpowder compositions, the active ingredient in finely divided form may beused in admixture with a larger-sized pharmaceutically-acceptable inertcarrier comprising particles having a size, for example, of up to 100micrometers in diameter. Suitable inert carriers include sugars such aslactose. Desirably, at least 95% by weight of the particles of theactive ingredient have an effective particle size in the range of 0.01to 10 micrometers. Alternatively, the composition may be pressurized andcontain a compressed gas, such as nitrogen or a liquified gaspropellant. The liquified propellant medium and indeed the totalcomposition are preferably such that the active ingredient does notdissolve therein to any substantial extent. The pressurized compositionmay also contain a surface-active agent, such as a liquid or solidnon-ionic surface-active agent or may be a solid anionic surface-activeagent. It is preferred to use the solid anionic surface-active agent inthe form of a sodium salt.

A further form of topical administration is to the eye, as for thetreatment of immune-mediated conditions of the eye such as autoimmunediseases, allergic or inflammatory conditions, and corneal transplants.The complex I inhibitor is delivered in a pharmaceutically acceptableophthalmic vehicle, such that the complex I inhibitor is maintained incontact with the ocular surface for a sufficient time period to allowthe complex I inhibitor to penetrate the corneal and internal regions ofthe eye, as for example the anterior chamber, posterior chamber,vitreous body, aqueous humor, vitreous humor, cornea, iris/cilary, lens,choroid/retina and sclera. The pharmaceutically acceptable ophthalmicvehicle may, for example, be an ointment, vegetable oil or anencapsulating material.

Complex I inhibitors may also be administered in the form of liposomes.As is known in the art, liposomes are generally derived fromphospholipids or other lipid substances. Liposomes are formed by mono-or multi-lamellar hydrated liquid crystals that are dispersed in anaqueous medium. Any non-toxic, physiologically acceptable andmetabolizable lipid capable of forming Liposomes can be used. Thepresent compositions in liposome form can contain, in addition to acomplex I inhibitor, stabilizers, preservatives, excipients, and thelike. The preferred lipids are the phospholipids and the phosphatidylcholines (lecithins), both natural and synthetic. Methods to formliposomes are known in the art. See, for example, Prescott, Ed., Methodsin Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p.33 et seq.

Another object of the present invention refers to a method of screeningfor a compound which inhibits complex I-mediated ROS production, themethod comprising

-   -   (a) contacting cells, particularly T-cells, with the compound        under investigation,    -   (b) staining said cells with an oxidation-sensitive dye, and    -   (c) measuring the intensity of said dye        wherein a decreasing intensity as compared to cells that are not        contacted with said compound is indicative of an inhibitory        effect of said compound.

Dyes that are sensitive to oxidation, i.e. can be used to determine ROSproduction, and as a result, change their intensity are well-known inthe art. In principle, all dyes which change their intensity upon anoxidation shift can be used for the method of the instant invention. Ina preferred embodiment, the dye to be used for the determination ofoxygen species is fluorescently labelled.

Examples of such dyes include, among others, Carboxy-H₂DCFDA, Milobox,CM-H₂DCFDA, Dihydrocalcein AM, Dihydrorhodamine 123, Dihydrorhodamine6G, H₂DCFDA, Lucigenin, Luminol, RedoxSensor Red CC-1,3′-(p-Aminophenyl)fluorescein, 3′-(p-Hydroxyphenyl) fluorescein, CM-H₂DCFDA, Proxylfluorescamine, TEMPO-9-AC, BODIPY FL EDA, BODIPY 665/676,Carboxy-H₂DCFDA, DPPP, cis-Parinaric acid, Singlet Oxygen Sensor Greenreagent, Coelenterazine, Dihydroethidium. Most preferably, H₂DCFDA isused as the dye.

If the dye is fluorescently labeled, the intensity is preferablymeasured by fluorescent-activated cell sorting (FACS) analysis or usinga fluorometer.

Examples of how to perform the method of invention is described inExample 8 or in Guelow et al (Journal of Immunol. 2005 May 1;174(9):5249-60).

As described supra in the present invention, the inflammatory diseaseswhich may be treated by the use of complex I inhibitors, respectivelythe medicaments containing them, may arise due to an increased ROSproduction.

Therefore, the present invention further refers to a method ofinhibiting ROS production within cells, the method comprising the stepof treating said cells with a complex I inhibitor.

DESCRIPTION OF THE DRAWING

FIG. 1. Activation-induced ROS generation depends on the proximal TCRsignalling machinery. A, B, Jurkat J16-145 cells, P116 (ZAP70 negativeJurkat cells), J.CaM2 (LAT negative Jurkat cells), J.CaM2/LAT(LAT-retransfected control cells), J14 (SLP76 deficient Jurkat cells),J14 76-11 (SLP76 retransfected control cells), J.γ1 (PLCγ1 deficientJurkat cells) and J.γ1/PLCγ1 (PLCγ1-retransfected control cells) werestimulated via plate-bound anti-CD3 antibodies (A) or with PMA (B) for30 min. Thereafter, cells were stained with DCFDA. Representative FACSprofiles for activation-induced DCFDA oxidation are shown. C, Schematicdiagram of TCR signalling.

FIG. 2. PKCθ is required for activation-induced generation of ROS. A,J16-145 cells were pretreated with the indicated amounts of the PKCinhibitor BIM and stimulated with PMA (left panel) or via plate-boundanti-CD3 antibodies (right panel) for 30 min. Cells were stained withDCFDA and analysed by FACS. Data shown as percentage of increase in MFI.B, J16-145 cells were pretreated with indicated amounts of general PKCpseudosubstrate peptide inhibitor, stained with DCFDA and stimulatedwith PMA (left panel) or via plate-bound anti-CD3 antibodies (rightpanel) for 30 min. ROS levels were measured as in (A). C, J16-145 Jurkatcells were pretreated with the indicated amounts of the PKC inhibitorBIM and stimulated with PMA/ionomycin (left panel) or via plate-boundanti-CD3 antibodies (right panel). After 1 h, RNA was isolated, reversetranscribed, and amplified using CD95L- and actin-specific primers. D,Jurkat J16-145 cells were transfected with 900 nM of scrambled- (ctr) orPKCδ-siRNA oligonucleotides (PKCδ). After 96 h, transfected cells werelysed, and analysed by Western blot for content of PKCδ (right panel) orstained with DCFDA, stimulated via PMA for 30 min, and subjected to FACSanalysis (left panel; results are shown as percentage of increase inMFI). E, Jurkat J16-145 cells were transfected with 900 nM of scrambled-(ctr) or PKCδ-siRNA oligonucleotides (PKCθ). 96 h after transfectioncells were analysed as described in (D) Western blot for PKCθ content(left panel); PMA-induced DCFDA oxidation (right panel). F, JurkatJ16-145 cells were pretreated with PKCθ pseudosubstrate peptideinhibitor and stimulated with PMA (left panel) or by plate-boundanti-CD3 antibodies (right panel) for 30 min. DCFDA oxidation wasmeasured by FACS and presented as increase of MFI. G, Jurkat cells weretransfected with scrambled- (ctr) or PKCθ-siRNA oligonucleotides (PKCθ)(as described in E). Cells were stimulated with PMA/ionomycin. After 1h, RNA was isolated, reverse transcribed, and amplified using CD95L- andactin-specific primers.

FIG. 3. Activation-induced ROS generation is partially NADPH oxidasedependent. A, B, Jurkat J16-145 cells were pretreated with NADPH oxidaseinhibitors DPI (A) and apocynin (B), stained with DCFDA and stimulatedwith PMA for 30 min. Inserts show neutrophils (NO) stimulated with PMA(10 ng/ml) and cotreated with DPI (100 μM) (A) or apocynin (600 μM) (B)to inhibit the NADPH oxidase dependent “oxidative burst”. Data arepresented as FACS-measured increase of MFI of oxidised DCFDA.

FIG. 4. PKCθ is translocated towards mitochondria upon PMA treatment. A,J16-145 cells were stimulated with PMA for 10 min. Cells were lysed andcellular fractions were separated as depicted in diagram (S1,S2—respective supernatants, P1, P2—respective pellets,). Highlightedfractions were separated by SDS-PAGE and analyzed by Western blot forcontent of PKCθ, PKCδ, ZnCuSOD (cytoplasmic marker), MnSOD(mitochondrial marker) and LAT (plasma membrane marker). B, Schematicdiagram of PKCθ translocation and ROS induction. C-H, Involvement ofmtDNA encoded proteins in activation-induced ROS generation and AICD. C,Total cellular DNA was isolated from parental J16-145 cells, J16-145cells cultured in the presence of uridine (50 μg/ml) and pyruvate (110mg/ml) (U+P) and J16-145 cells cultured in the presence of U+P andethidium bromide (250 ng/ml) (ps-ρ⁰). For PCR amplification of theorigin of replication of mitochondrial heavy strand (mt-ori), 100 ng ofDNA template was used (upper panel). Amplification of the β-actin genefragment was used as a loading control (lower panel). D, Cells depletedof mtDNA show an impaired activation-induced ROS. Parental J16-145 cellscultured in medium supplemented with U+P (U+P) or cells depleted ofmtDNA)(ps-ρ⁰) were stimulated via plate-bound anti-CD3 antibodies (leftpanel) or with PMA (right panel) for 30 min, stained with DCFDA andanalysed by FACS. The percentage of increase in MFI is shown. E, Cellsdepleted of mtDNA show lowered AICD. Cells (U+P) or (ps-ρ⁰) as describedin (C) were stimulated via plate-bound anti-CD3 antibodies or withPMA/ionomycin. After 24 h cell death was measured by a drop in FSC/SSCindex and results were recalculated to “specific cell death”. F, Thecontent of mtDNA was tested for parental J16-145 cells (J16-145) orps-ρ⁰ cells, which regained mtDNA after long-term culture due towithdrawal of ethidium bromide from culture medium (recov.). Totalcellular DNA (100 ng) was used and amplified as described in (C). G,Parental Jurkat J16-145 cells cultured in standard medium (J16-145) orps-ρ⁰ cells after recovery of mtDNA content (recov.) were stimulated byplate-bound anti-CD3 antibodies (left panel) or with PMA (right panel)for 30 min, activation-induced ROS production was measured as in (D). H,Parental J16-145 (J16-145) or (recov.) cells were stimulated byplate-bound anti-CD3 antibodies (left panel) or with PMA/ionomycin(right panel). After 24 h cell death was determined as described in (E).

FIG. 5. Complex I of the mitochondrial ETC is the source ofactivation-induced ROS formation A, B, Jurkat J16-145 cells werepretreated with the indicated amounts of ETC inhibitors (ROT—rotenone;Pier—piericidin A; AA—antimycin A; Az—sodium azide) or an inhibitor ofthe F0F1-ATPase (OLI—oligomycin), stained with DCFDA and stimulated byPMA (A) or via plate-bound anti-CD3 antibody (B) for 30 min and analysedby FACS. The data are presented as percentage of increase in MFI. C,Jurkat J16-145 cells were treated with high concentrations of inhibitorsof the ETC or the F0F1-ATPase for 2 h. Thereafter, cells were lysed andATP content was determined. D, Mitochondria-derived ROS induce changesin expression and activity of MnSOD. Jurkat J16-145 cells werestimulated with plate bound anti-CD3 antibodies or PMA/ionomycin (Iono)for the indicated time period. Isolated RNA was reverse-transcribed, andamplified using MnSOD-specific primers. E, Jurkat cells were stimulatedvia plate bound anti-CD3 antibodies or PMA/ionomycin (Iono) for theindicated time points. Cells were lysed and MnSOD protein levels weredetermined by Western blot analysis. MnSOD expression was normalized totubulin and quantified using NIH Image (lower panel). F, MnSOD activityin mitochondria of J16-145 cells stimulated by plate-bound anti-CD3antibody or PMA.

FIG. 6. ROS produced by complex I drive activation-induced CD95Lexpression A, B, J16-145 cells were pretreated with the indicatedamounts of inhibitors of the ETC (ROT—rotenone; Pier—piericidin A;AA—antimycin A; Az—sodium azide) or the F0F1-ATPase (OLI—oligomycin) andstimulated with PMA/ionomycin (Iono) (A) or plate-bound anti-CD3antibodies (B) for 1 h. RNA was isolated, reverse-transcribed, andamplified using CD95L- and actin-specific primers. C, J16-145 cells werepretreated with the indicated inhibitors and stimulated with (leftpanel) or without (right panel) plate-bound anti-CD3 antibodies for 1 h.RNA was isolated, reverse-transcribed, and a quantitative PCR wasperformed. CD3 induced CD95L expression was set to 100%. All othervalues were calculated according to the CD3 induced CD95L expression. D,Schematic diagram of mitochondrial ROS production. C_(I)=complex I.

FIG. 7. Downregulation of NDUFAF1 inhibits ROS generation, CD95Lexpression and AICD. A, J16-145 cells were transfected with 75 nMscrambled- (ctr) or two different NDUFAF1-siRNA oligonucleotides (#1,#2). After 48 h RNA was isolated, reverse-transcribed, and amplifiedusing NDUFAF1- and actin-specific primers. B, 48 h after transfectionwith scrambled- (ctr) or NDUFAF1-siRNA oligonucleotides (#1, #2) theoxidative signal upon 30 min of PMA treatment was determined by DCFDAstaining (filled profile—stained cells/untreated; open profile—cellsstained and stimulated with PMA). C, Quantification of PMA-inducedoxidative signals in Jurkat cells. 72 h after transfection with 75 nMscrambled- (ctr) or NDUFAF1-siRNA oligonucleotides (#1, #2). Cells werestained with DCFDA, treated with PMA for 30 min and subjected to FACSanalysis. Results are shown as percentage of increase in MFI. D, J16-145cells were transfected with 75 nM of scrambled- (ctr) or NDUFAF1-siRNAoligonucleotides (#1, #2). After 72 h of resting, cells were treatedwith PMA/ionomycin for 1 h. PMA/ionomycin RNA was isolated,reverse-transcribed, and amplified using CD95L-, and actin-specificprimers. E, J16-145 cells were transfected with 75 nM (left panel) or900 nM (right panel) of scrambled- (ctr) or NDUFAF1-siRNA #2oligonucleotides. After 72 h of resting AICD was induced by 24 h ofPMA/ionomycin treatment. Cell death was assessed by a drop in FSC/SSCindex. Results were recalculated to “specific cell death”.

FIG. 8. Metformin, a non-toxic complex I inhibitor, blocksactivation-induced oxidative signal, CD95L expression and AICD. A,Metformin induces no toxicity. J16-145 cells were treated for 2 h (whitebars) and 24 h (grey bars) with the indicated inhibitors. Cell death wasdetermined by a drop in forward-to-side-scatter profile in comparison toliving cells and recalculate to “specific cell death”. (ROT—rotenone [10μg/ml]; Pier—piericidin A [7.5 μM]; Metf—metformin [100 μM];TTFA—1,1,1-thenoyl trifluoroacetone [25 μM]; AA—antimycin A [4 μg/ml];Az—sodium azide [100 μg/ml]; OLI—oligomycin[10 μg/ml]). B, J16-145 cellswere pretreated with indicated amounts of metformin, stained with DCFDAand stimulated by PMA for 30 min. Oxidative signal was quantified asincrease in MFI. C, D, J16-145 cells were pretreated with the indicatedamounts of metformin and stimulated with PMA/ionomycin (Iono) (C) orplate-bound anti-CD3 antibodies (D) for 1 h. Next, RNA was isolated,reverse-transcribed, and amplified using CD95L- and actin-specificprimers (left panel). In addition, a quantitative PCR was performed(right panel). CD3 induced CD95L expression was set to 100%. All othervalues were calculated according to the CD3 induced CD95L expression. E,F, J16-145 cells pretreated with the indicated amounts of metformin andAICD was induced by PMA/ionomycin (Iono) treatment (E) or stimulationwith plate-bound anti-CD3 antibodies (F). After 24 h cell death wasassessed by drop in FSC/SSC index. Inserts (E, F) show J16-145 cellscotreated with or without CD95L neutralizing antibody and stimulated byplate-bound anti-CD3 antibodies. Results were recalculated to “specificcell death”.

FIG. 9. Primary human T cells depend on complex I-originatedactivation-induced ROS for CD95L expression and AICD. A, T cells werepretreated with the indicated amounts of inhibitors of the ETC(ROT—rotenone and AA—antimycin A) and stimulated with anti-CD3antibodies for 30 min. Cells were stained with DCFDA and MFI wasmeasured by FACS. B, T cells were pretreated with indicated amounts ofrotenone (upper panel) or antimycin A (lower panel) and stimulated withanti-CD3 antibodies for 1 h. Next, RNA was isolated,reverse-transcribed, and amplified using CD95L- and actin-specificprimers. C, T cells were transfected with 900 nM of scrambled- (ctr) ortwo different NDUFAF1-siRNA oligonucleotides (#1, #2). After 48 h, RNAwas isolated, reverse-transcribed, and amplified using NDUFAF1- andactin-specific primers. D, 72 h after transfection with 900 nM ofscrambled- (ctr) or NDUFAF1-siRNA oligonucleotides (#1, #2) primaryhuman T were stimulated by plate-bound anti-CD3 antibodies for 30 minand oxidative signal was determined as in (A). E, F, G, The T cells werepretreated with indicated amounts the non-toxic complex I inhibitor,metformin, and stimulated with plate-bound anti-CD3 antibodies (i) for30 min (E), to measure the oxidative signal (quantified as in (A)). (ii)For 1 h (F), to detect changes in CD95L expression (left panel semiquantitative PCR; right panel quantitative PCR). (iii) For 24 h (G) toassessed AICD by a drop in FCS/SSC index. Insert show T cells cotreatedwith or without CD95L neutralizing antibody (results were recalculatedto “specific cell death”). H, Schematic diagram of TCR induced oxidativesignalling.

FIG. 10. A and B, Non-activated (day “0”—A) or pre-activated (day “6”—B)peripheral human T cells were pre-treated with indicated amounts ofrotenone for 5 min and stimulated with anti-CD3 antibodies for 1 h.Next, RNA was isolated, reverse-transcribed, and amplified using IL-2,IL-4, TNF α, IL-13 and actin-specific primers.

C, J16-145 Jurkat T cells were transfected with 75 nM of scrambled-(ctr) or NDUFAF1-siRNA oligonucleotides (oligonucleotides #1 and #2).After 72 h of resting, cells were treated with PMA/ionomycin for 1 h.Subsequently, RNA was isolated, reverse-transcribed, and amplified usingIL-2, IL-4 and actin-specific primers.

D, Pre-activated (day “6”) peripheral human T cells were pre-treatedwith indicated amounts of metformin for 1 h. Next, cells were stimulatedwith anti-CD3 antibodies for 1 h. Subsequently, RNA was isolated,reverse-transcribed, and amplified using TNF α and actin-specificprimers.

The invention is further explained by the following examples withoutbeing bound to it.

EXAMPLES Example 1

Chemicals. Dichlorodihydrofluorescein diacetate (DCFDA) was obtainedfrom Molecular Probes, Germany. The cell permeable, myristoylatedpseudosubstrate peptide inhibitors (general anti-PKC and anti-PKCθ) werepurchased from Calbiochem, Germany. Primary antibodies against humanPKCδ and PKCθ were supplied by BD Transduction Lab., Germany. Primaryantibodies against human MnSOD and LAT were obtained from UpstateBiotech., USA. The primary antibody against human ZnCuSOD was purchasedfrom Santa Cruz Biotech., Germany. The neutralising anti-CD95L antibodyNok1 was obtained from BD Pharmingen, Germany. All other chemicals andprimary antibodies against human tubulin α were supplied bySigma-Aldrich, Germany. The agonistic monoclonal antibody anti-Apo-1(mouse IgG3) recognizing an extracellular part of CD95 (Apo1/Fas) (62)and the monoclonal anti-CD3 antibody OKT3 (25) were prepared asdescribed.

Example 2

Cell culture. Jurkat J16-145 is a sub-clone of the human Tlymphoblastoid cell line Jurkat J16 (25). J.CaM2 is a LAT negativeJurkat cell line and J.CaM2/LAT is the control cell line retransfectedwith LAT (19). P116 is a ZAP70 negative Jurkat cell line (68) andP116cl.39 is the retransfected control cell line. J14 is a SLP76deficient cell line and J14 76-11 is the retransfected control cell line(37). J.γ1 is a PLCγ1 deficient Jurkat cell line and J.γ1/PLCγ1 is theretransfected control cell line (29). Jurkat cells were cultured in IMDMmedium supplemented with 10% FCS.

Example 3

Generation of pseudo-ρ⁰ cells. Cells depleted of mitochondrial DNA(mtDNA) were generated as described previously (12, 36) with minormodifications. Briefly, Jurkat J16-145 cells were cultured in IMDMmedium supplemented with ethidium bromide (250 ng/ml) for up to 21 days.Ethidium bromide accumulates in much higher concentrations in themitochondrial matrix than in the nucleus. Therefore, it can be used toselectively inhibit mtDNA replication. The amount of mtDNA was examinedby isolation of DNA followed by PCR specific for the mitochondrialorigin of replication (ori-mt).

The amplified product spanned ori-mt of the mtDNA heavy strain betweenposition 15868 and 754: sense 5′-GAAAACAAAATACTCAAATGGGCC-3′ andantisense 5′-CCTTTTGATCGTGGTGATTTAGAGGG-3′. Cells depleted in mtDNA relyenergetically mainly on glycolysis and have impaired nucleotidemetabolism. Therefore, pseudo-ρ⁰ cells were further cultured in IMDMmedium supplemented with ethidium bromide (250 ng/ml), uridine (50μg/ml), and sodium pyruvate (110 mg/ml). Since cells were not completelydeficient in mtDND they will be referred as pseudo-ρ⁰ cells (12). Toreconstitute mtDNA content pseudo-ρ⁰ cells were transferred to thestandard medium. Cells recovered to normal phenotype in 21-23 days.

Example 4

Isolation of total cellular DNA. Jurkat J16-145 cells were lysed for 1 hat 55° C. in 0.2M sodium acetate, 6.25% SDS solution containing 250μg/ml proteinase K. Genomic DNA was isolated by aphenol/chloroform-extraction.

Example 5

Isolation of human peripheral T cells. Human peripheral T cells wereprepared by Ficoll-Plaque density centrifugation, followed by rosettingwith 2-amino-ethylisothyo-uronium-bromide-treated sheep red blood cellsas described (25). For activation, resting T cells were cultured at aconcentration of 2×10⁶ cells/ml with 1 μg/ml PHA for 16 h. Next, T cellswere cultured in RPMI 1640 supplemented with 10% FCS and 25 U/ml IL-2for 6 days (day 6 T cells) as described (25).

Example 6

Isolation of human polymorphonuclear cells. Neutrophils from healthyindividuals were prepared by Polymorphprep® density centrifugationaccording to the manufacturer's instructions (Axis-Shield, Norway).

Example 7

Assessment of cell death. To induce CD95L expression and/or subsequentapoptosis, cells were stimulated with anti-CD3 antibody (OKT3, 30 μg/ml)or PMA (10 ng/ml) and ionomycin (1 μM). Cell death was assessed by adrop in the forward-to-side-scatter (FSC/SSC) profile in comparison toliving cells and recalculated to “specific cell death” as described(25).

Example 8

Determination of anti-CD3 and PMA induced ROS generation. Jurkat cellswere stimulated either with plate-bound anti-CD3 (OKT3, 30 μg/ml) or byPMA (10 ng/ml) for 30 min and stained with the oxidation-sensitive dyeH₂DCFDA (5 μM). Since activation-induced ROS generation in human T cellswas maximal at 30 min stimulation (25) this time point was chosen forall experiments. Incubation was terminated by washing with ice-cold PBS.ROS generation was determined by FACS and quantified as “Increase inMean Fluorescence Intensity (MFI)” [%], calculated according to thefollowing formula: “Increase in MFI”[%]=[(MFI_((stimulated))−MFI_((unstimulated)))/MFI_((unstimulated))]×100as described (15). Cells were preincubated with inhibitors for 5 minprior to stimulation with exception of anti-PKC peptide inhibitors (20min) and metformin (1 h). All experiments were performed in triplicates.Results shown are representative of at least three independentexperiments.

Example 9

ATP determination. Cells were lysed by freezing and thawing. CellularATP was measured according to manufacturer's instructions(ATP-determination Kit, Molecular Probes, Germany).

Example 10

Mitochondria isolation and Western blot analysis. Crude mitochondrialfraction (containing membrane impurities) and cytoplasmic fraction wereisolated using Mitochondria Isolation Kit (Pierce, USA) according to themanufacturer's instructions. Next, membranes were separated frommitochondria by isopycnic 0.8-2M sucrose gradient centrifugation for 2 hat 80 000 g. FIG. 4A shows schematic diagram of the purificationprocedure. Cells were lysed in RIPA lysis buffer [60 mM NaCl, 25 mMTris/HCl, 0.5% desoxycholate, 1 mM DTT and Halt Protease InhibitorCocktail (Pierce, USA)] and protein concentration was measured by BCAassay (Pierce, USA). SDS-PAGE and Western blot analysis was performed asdescribed (24). Western blots were quantified by standard scanningdensitometry using the NIH Image program version 1.36b.

Example 11

RNA preparation and semi-quantitative RT-PCR. RNA was isolated usingTrizol reagent (Invitrogen, Germany) according to manufacturer'sinstructions. Total RNA (5 μg) was reverse-transcribed using a RT-PCRkit (Applied Biosystems, Germany). Aliquots were amplified by PCR asdescribed (25). Primers for detection of CD95L, β-actin and NDUFAF1 wereused as described (25, 40, 66). Primers used for amplification of MnSOD(SOD2) and Zn/CuSOD (SOD1) transcripts were: MnSOD, sense5′-CTTCAGCCTGCACTGAAGTTCAAT-3′ antisense 5′-CTGAAGGTAGTAAGCGTGCTCCC-3′and Zn/CuSOD, sense 5′-GCGACGAAGGCCGTGTGCGTGC-3′ antisense5′-CTAGAATTTGCGGTGGACGATGGAGGG-3′.

Example 12

Quantitative PCR. The primers and fluorescent-labeled probes used herewere CD95L sense 5′-AAAGTGGCCCATTTAACAGGC-3′, antisense5′-AAAGCAGGACAATTCCATAGGTG-3′, probe 5′-TCCAACTCAAGGTCCATGCCTCTGG-3′;β-actin sense 5′-ACCCACACTGTGCCCATCTACGA-3′, antisense5′-CAGCGGAACCGCTCATTGCCAATGG-3′, probe 5′-ATGCCCTCCCCCATGCCATCCTGCGT-3′.PCR reaction mixture (PCR kit from Eurogentech, Belgium) contained 80 μgof reverse-transcribed cDNA, 1.25±7.5 pM forward primers, 22.5 pMreverse primers and 5 pM probe. For each sample three PCRs wereperformed. The resulting relative increase in reporter fluorescent dyeemission was monitored by the TagMan-system (GeneAmp 5700 sequencedetection system and software, Perkin Elmer, Foster City, Calif., USA).The level of the CD95L and CD95 mRNA, relative to β-actin mRNA wascalculated using the formula: Relative mRNAexpression=2^(−(Ct of CD95L−Ct of b-actin)), where Ct is the thresholdcycle value.

Example 13

Transfection and siRNA-mediated knock down. Jurkat T cells and primaryhuman T cells were transfected by lipofection (HiPerfect, Qiagen,Germany) with negative control siRNA oligonucleotides (unlabeled orAlexa 488 labeled non-silencing siRNA, Qiagen, Germany), siRNAoligonucleotides specific for human NDUFAF1: oligo#1 antisense strand:5′-ACUAACAUCAGGCUUCUCCdTdT-3′, oligo#2 antisense strand:5′-UAACUAUACAUCUGAUUCGdTdT-3′ or siRNA oligonucleotides specific forhuman PKCδ (Hs_PKKCD_(—)11_HP) and PKCθ (Hs_PRKCQ_(—)5_HP) (Qiagen,Germany). Transfection was performed using 2×10⁵ cells, 9 μl oftransfection reagent and different amounts of siRNA oligonucleotidesranging from 75 nM to 900 nM according to manufacturer's instructions.Transfected cells were rested for 48 h before being subjected to furtherexperiments.

Example 14

Measurement of MnSOD activity. MnSOD activity was determined using acommercial kit (Dojindo Molecular Technologies, Japan). 1.5×10⁷ cellswere stimulated by plate-bound anti-CD3 antibody (OKT3, 30 μg/ml) orwith PMA (10 ng/ml) for different time periods. Cells were harvested andlysed by freezing and thawing. Protein content was adjusted to 1 mg/mland SOD activity was measured with a photometer according to themanufacturer's instructions. MnSOD activity was assessed after blockingbackground activity of ZnCuSOD by addition of 1 mM KCN to the reactionmixture.

Example 15

TCR-Induced ROS Generation Depends on the Proximal TCR SignallingMachinery.

Previous studies indicate that TCR stimulation leads to generation of anoxidative signal involving H₂O₂ (15, 25). This H₂O₂ signal is vital forthe initiation of CD95L promoter activity, CD95L expression and AICD(25). In order to identify the components of the transduction cascademediating ROS generation, we used cells deficient in TCR-signallingmolecules. Jurkat cell lines deficient in ZAP70 (68), Lat (19), SLP76(37) and PLCγ1 (29) were stained with DCFDA and stimulated withplate-bound anti-CD3 antibodies for 30 minutes. All deficient cell linesdid not display any oxidative signal, whereas retransfected controlsshowed a clear increase of ROS upon TCR stimulation (FIG. 1A). Thus, weconclude that TCR-induced generation of ROS depends on ZAP70, Lat andPLCγ1 (FIG. 1C). As PLCγ1 activation results in triggering of PKCs weinvestigated a possible role of PKCs in oxidative signalling by treatingthe deficient cell lines with PMA, a PKC activator, which by-passesZAP70, Lat, SLP76 and PLCγ1. Remarkably, all deficient cell linesrevealed a PMA-induced oxidative signal (FIG. 1B). PMA induces anoxidative signal without influencing the intracellular Ca²⁺ level (25).This implicates an involvement of Ca²⁺-independent PKCs in TCR-inducedoxidative signalling (FIG. 1C).

Example 16

PKCθ is required for activation-induced generation of ROS.

To further corroborate an involvement of PKCs in activation-induced ROSformation cells stimulated via PMA and plate-bound anti-CD3 antibodieswere pretreated with the general PKC inhibitor bisindolylmaleimide I(BIM) (FIG. 2A) or a PKC-specific peptide inhibitor (FIG. 2B). Bothinhibitors blocked more than 95% of the oxidative signal. Since ROScooperates with Ca²⁺ signalling for CD95L induction (25) we analysed theimpact of BIM on CD95L expression. Cells stimulated via anti-CD3antibodies and PMA/ionomycin were pretreated with BIM. RNA was isolated,reverse-transcribed, and amplified using CD95L specific primers. In BIMtreated cells a dose dependent inhibition of CD95L expression wasdetectable (FIG. 2C). Considering that activation-induced oxidativesignalling is inducible by PMA alone (FIG. 1B), we focused onCa²⁺-independent novel PKC isoforms (nPKC). It has been reported thatPKCδ, a nPKC isoform, is involved in ROS generation in keratinocytesupon overexpression (39) and in PMA/ionomycin treated myeloid leukemiacells (43). However, despite downmodulating PKCδ via siRNA, theoxidative signal was not significantly affected (<20% decrease) (FIG.2D). This implies that PKCδ only plays a minor role in generation ofactivation-induced ROS. PKCθ is unique among the nPKC isoforms becauseit is indispensable for T cell development and activation (48, 59, 64).To analyze the impact of PKCθ on activation-induced ROS production,cells were transfected with PKCθ siRNA oligonucleotides (FIG. 2E). Morethan 80% of the PMA-induced oxidative signal was inhibited in cellstransfected with PKCθ siRNA oligonucleotides as compared to the control.These data were further confirmed by treating Jurkat cells with aPKCθ-specific peptide inhibitor, which significantly reduced the TCR-and the PMA-induced ROS levels (FIG. 2F). Moreover, siRNA mediateddownmodulation of PKCθ results in an inhibition of CD95L expression(FIG. 2G). Thus, we conclude that PKCθ is crucial for activation-inducedROS formation and CD95L expression.

Example 17

Activation-Induced ROS Generation is Partially NADPH Oxidase Dependent.

Recently, it has been shown in NOX2 deficient mice that TCR-induced ROSgeneration is, at least partially, dependent on NOX2 (30). Here weanalysed a potential role of NADPH oxidases in human T cells. Jurkatcells were preincubated with DPI, a rather unspecific but commonly usedNADPH oxidase inhibitor (14, 42, 55, 57, 61) or the specific NADPHoxidase inhibitor apocynin (60) and thereafter treated with PMA as anNADPH oxidase activator. Application of both, DPI and apocynin showedonly a moderate effect on PMA-induced ROS generation in Jurkat cells(FIG. 3A, B). To control whether the applied amounts of inhibitors aresufficient to block NADPH oxidase, neutrophils were treated with PMA (10ng/ml) and cotreated with DPI and apocynin. PMA induces a massive NADPHoxidase dependent ROS release in neutrophils called “oxidative burst”.The “oxidative burst” could be inhibited almost completely (up to 91%inhibition) by application of 100 μM DPI or 600 μM apocynin. The sameamounts of inhibitors block in Jurkat cells less than 60% of thePMA-induced oxidative signal (FIG. 3A, B). Since downmodulation of PKCθexpression inhibited more than 80% of the oxidative signal (FIG. 2E),the existence of an additional PKCθ-dependent source of ROS in human Tcells has to be postulated.

Example 18

Cells Depleted in Mitochondrial DNA Show an Impaired Activation-InducedROS Generation and AICD.

Besides NOX2, mitochondria are a prominent source of ROS. It has beenreported that upon PMA treatment PKCδ can be translocated into/tomitochondria (39, 43). To determine whether PKCθ is translocated tomitochondria Jurkat cells were stimulated with PMA. PKC translocationwas assessed by subjecting cytoplasmic, mitochondrial and plasmamembrane fractions to immunoblotting with anti-PKC antibodies.Surprisingly, PKCδ and PKCθ were detected in the plasma membranefraction already in unstimulated cells. However, as expected, PKCδtranslocates to the mitochondria after stimulation. Interestingly, theamount of PKCθ increases also in the mitochondrial fraction upon PMAtreatment (FIG. 4A). Thus, PKCθ and PKCδ are translocated to themitochondria and/or associated membranes in T cells after activation(FIG. 4B). In order to analyse the role of mitochondria inactivation-induced ROS generation in more detail, cells transientlydepleted in mtDNA, pseudo-ρ⁰ cells, were generated by short exposure(6-21 days) to low amounts of ethidium bromide (FIG. 4C) (12, 36). Uponstimulation with anti-CD3 or PMA, pseudo-ρ⁰ cells exhibited an up to 60%diminished oxidative signal (FIG. 4D). Since the activation-inducedoxidative signal is crucial for AICD pseudo-ρ⁰ cells displayed a massivereduction of AICD upon TCR-stimulation and PMA/ionomycin treatment (FIG.4E). The depletion of mtDNA was entirely reversible after removal ofethidium bromide from cell culture for 21-23 days (FIG. 4F). Inconcordance with recovery of mitochondrial protein expression,activation-induced ROS generation (FIG. 4G) and AICD (FIG. 4H) regainedits normal level. Thus, we demonstrate here that mitochondrial functionis a prerequisite for induction of AICD.

Example 19

Complex I of the Mitochondrial ETC is the Source of Activation-InducedROS Formation.

Since depletion of mtDNA leads to a decrease in activation-induced ROSgeneration, mtDNA-encoded proteins have to be involved in oxidativesignalling. Most enzymes of the ETC are oligomeric complexes consistingof both nuclear DNA- and mtDNA-encoded subunits. The primary sites formitochondrial ROS production are the complexes I and III of the ETC(45). Therefore, we aimed at analysing the role of these complexes inactivation-induced ROS production. Complex I was blocked by rotenone, acommonly used inhibitor. However, rotenone is also known to interferewith a couple of cellular pathways including tubulin-depended signallingevents (8, 16, 32, 52). Thus, we also used a second more specificinhibitor, namely piericidin A (13, 28). Complex II was inhibited byapplication of 1,1,1-thenoyl trifluoroacetone (TTFA), complex III byantimycin A, complex IV by sodium azide and the F₀F₁ ATPase was blockedby oligomycin. Cells were pretreated with these inhibitors andsubsequently stimulated with anti-CD3 antibodies or PMA. Thereafter,generation of ROS was determined. Only rotenone and piericidin A wereable to inhibit activation-induced ROS generation, whereas TTFA,antimycin A, sodium azide, and oligomycin had no effect or increased theoxidative signal (FIG. 5A, B). ATP levels could not account forinhibition of ROS generation, since oligomycin and antimycin A treatmentresulted in a more efficient ATP depletion as compared to rotenone andpiericidin A (FIG. 5C). In contrast to inhibition of the NADPH oxidase(max. 60% blockage of ROS generation; FIG. 3), inhibition of complex Ileads to a blockage of more than 95% of activation-induced ROSproduction (FIG. 5A, B). Therefore, complex I is not only the source ofmitochondria-derived ROS but also its activity seems to be aprerequisite for subsequent ROS production via the NADPH oxidase.

Example 20

Activation-Induced ROS Enhances Expression and Activity of MitochondrialMnSOD.

It has been demonstrated that complex I releases superoxide anion (O₂ ⁻)into the mitochondrial matrix (65). However, it is cytosolic H₂O₂ whichplays a crucial role in CD95L expression (25). Since O₂ ⁻ cannot crossmembranes (53) and leave the mitochondria it has to be converted tomembrane permeable H₂O₂ by MnSOD to act as a second messenger ininduction of AICD. To analyse whether TCR stimulation and PMA treatmentresults in an upregulation of MnSOD transcription, Jurkat cells werestimulated with plate-bound anti-CD3 antibodies or treated withPMA/ionomycin. RNA was isolated and reverse-transcribed. After 60 min amoderate increase in the transcript level of MnSOD was detected inCD3-stimulated and PMA/ionomycin-treated cells, whereas cytosolicZnCuSOD level remained unchanged (FIG. 5D). In addition, induction ofMnSOD on protein level was analysed. Cells were stimulated with anti-CD3antibodies or PMA/ionomycin and lysed at the indicated time points.After 4 hours of stimulation an increase in MnSOD protein level wasobserved (FIG. 5E). To verify these data, activity of MnSOD wasdetermined. PMA treatment and CD3 stimulation led to a fast increase ofMnSOD activity (FIG. 5F). Thus, complex I derived ROS is transformed toH₂O₂ and therefore, it can serve as a second messenger in regulation ofCD95L expression (25) (FIG. 6D).

Example 21

Complex I Derived ROS are Crucial for Induction of CD95L Expression.

To analyse the role of complex I derived ROS in activation-induced CD95Lexpression we inhibited all components of the ETC. Cells were stimulatedby CD3 triggering (FIG. 6A) or PMA/ionomycin treatment (FIG. 6B) in thepresence or absence of inhibitors. After 1 hour of treatment RNA wasisolated, reverse-transcribed and amplified using CD95L-specificprimers. Significant levels of CD95L transcripts were not detected inunstimulated cells, whereas CD3 triggering and PMA/ionomycin (FIG. 6A,B) stimulation resulted in a strong expression of CD95L. The complex Iinhibitors, rotenone and piericidin A, abolished CD95L transcriptionalmost completely, whereas blocking the other complexes of the ETC hadno effect (FIG. 6A, B). To verify these data a quantitative PCR wasperformed. Stimulation of Jurkat cells with anti-CD3 antibodiesdisplayed a strong induction of CD95L expression. Rotenone treatmentresulted in a more than 80% reduction of CD95L induction, whereasantimycin A and oligomycin showed no effect (FIG. 6 C). Upon 2 h oftreatment, applied doses of all inhibitors were in a sub-toxic range(FIG. 8A), thus their toxicity can not account for inhibition ofactivation-induced ROS production and CD95L expression. Therefore, ROSgenerated from complex I are a prerequisite for induction of CD95Lexpression (FIG. 6D).

Example 22

siRNA-Mediated Downregulation of NDUFAF1 Expression InhibitsActivation-Induced ROS Signalling, CD95L Expression and AICD.

In addition to pharmacological manipulation of mitochondrialrespiration, we sought for other ways to abolish complex I function andinhibit activation-induced ROS signalling. Mammalian complex I consistsof at least 46 subunits (10). 39 of them are encoded by nuclear DNA andmay be a potential target for siRNA-mediated downregulation. Recently,it has been shown that NDUFAF1, a human homologue of CIA30—a complex Ichaperone of Neurospora crassa—is essential for assembly of complex I inhumans (31, 66). Reducing the amount of NDUFAF1 by siRNA led to alowered abundance and activity of complex I (66). To knock down NDUFAF1expression, we used two different siRNA oligonucleotides (66). BothsiRNAs displayed a knock down effect with siRNA oligonucleotide #2mediating a stronger inhibition of NDUFAF1 expression (FIG. 7A).Remarkably, both oligonucleotides diminished the oxidative signalinduced by PMA (oligonucleotide #1 up to 39% and oligonucleotide #2 upto 68%) (FIG. 7B, C). Since the oxidative signal generated by complex Iis required for CD95L expression (FIG. 6A, B), downregulation of theNDUFAF1 level has to influence transcription of CD95L. Jurkat cellstransfected with NDUFAF1 siRNA oligonucleotides were stimulated withPMA/ionomycin. After 1 h of treatment RNA was isolated,reverse-transcribed and amplified using CD95L-specific primers. Cellstransfected with control oligonucleotides showed a normal expression ofCD95L, whereas cells transfected with NDUFAF1 siRNA displayed a stronglydiminished CD95L expression (FIG. 7D). In addition, the role of complexI in AICD was analysed in cells transfected with NDUFAF1 siRNAoligonucleotides. AICD was determined after 24 h of PMA/ionomycintreatment. In comparison to control cells, cells transfected withNDUFAF1 siRNA oligonucleotides displayed a significant inhibition ofcell death (FIG. 7E). Thus, we prove that complex I assembly and ROSformation are crucial for AICD induction.

Example 23

Metformin Inhibits Complex I Derived ROS, Activation-Induced CD95LExpression, and AICD.

In search for potential tools to manipulate ROS generation at complex Iand treat CD95/CD95L-dependent disorders, we applied metformin, a drugwidely used in treatment of type II diabetes (2, 3, 58). Metformin hasrecently received attention due to its effects on mitochondria (6, 18,23). It has been demonstrated, that metformin mildly inhibits complex I(6, 18). In addition, metformin can efficiently inhibit complexI-induced ROS production in isolated mitochondria, an effect that waslinked to blockage of reversed electron flux (6). Since metformin showedno toxicity on Jurkat cells (FIG. 8A) it is an ideal tool to investigatethe impact of complex I-mediated ROS production on AICD. Jurkat cellspretreated with metformin and stained with DCFDA exhibited a diminishedoxidative signal after treatment with PMA (FIG. 8B). Concordantly, theyalso displayed an inhibition of CD95L expression upon PMA/ionomycintreatment and TCR triggering (FIG. 8C, D). A quantitative PCR revealedthat cells stimulated with PMA/ionomycin and cotreated with metformindisplayed a 50% reduction of CD95L expression (FIG. 8C). Even moreremarkably, upon TCR triggering metformin inhibits CD95L expression upto 80% (FIG. 8D). AICD in Jurkat cells is mainly CD95L dependent (FIG.8E, F). To study the effect of metformin on AICD, Jurkat cells wereeither stimulated with PMA/ionomycin (FIG. 8E) or plate-bound anti-CD3antibodies (FIG. 8F). Cells pretreated with metformin showed adrastically reduced AICD. Apoptosis induced via direct stimulation ofthe CD95 receptor was not affected by metformin (data not shown). Thus,blockage of AICD by metformin is due to inhibition of CD95L expression.

Example 24

Inhibition of Complex I Blocks AICD in Primary Human T Cells.

To further underline the physiological relevance of complex I derivedROS in AICD preactivated primary human T cells (day 6 T cells) wererestimulated with anti-CD3 antibodies and pretreated with or withoutrotenone or antimycin A. The activation-induced oxidative signal (FIG.9A) and CD95L expression (FIG. 9B) were exclusively inhibited byrotenone. In order to prove that complex I is the source of theoxidative signal, preactivated T cells (day 6 T cells) transfected withNDUFAF1 siRNA oligonucleotides (FIG. 9C) were used to measure ROSgeneration upon CD3 triggering. The NDUFAF1 siRNA oligonucleotidesabolished activation-induced ROS generation up to 30% (FIG. 9D). Inaddition, we analysed the effects of metformin on primary human T cells.Metformin inhibits the anti-CD3 induced oxidative signal (FIG. 9E) andinduction of CD95L expression (up to 70% inhibition) (FIG. 9F). SinceAICD is mainly CD95L dependent, cell death was nearly completelyinhibited by metformin (FIG. 9G). Apoptosis in primary T cells inducedby direct stimulation of the CD95 receptor was not affected by metformintreatment (data not shown). Thus, the non-toxic complex I inhibitorseems to be a promising tool for treatment of diseases wherederegulation of CD95L expression plays a crucial role.

Discussion

The molecular source and the signalling steps necessary for ROSproduction are largely unknown. Here we show for the first time thatactivation-induced ROS generation depends on the classical components ofthe TCR signalling machinery (FIG. 9H). Upon TCR stimulation Lat isphosphorylated by ZAP70 and recruits PLCγ1, which generates IP₃ and DAG.DAG, as well as its mimetic PMA, activates several classes of enzymes,namely PKCs, PKDs, DGKs, RasGRP and chimearins (9). However, we showedhere an involvement of PKCs in activation-induced oxidative signalling.Application of BIM, a PKC-ATP binding blocker, and a specificpseudosubstrate peptide inhibited PMA- and TCR-induced ROS generation.PMA induces an oxidative signal without influencing the cytosolic Ca²⁺level (25). Therefore, it is probable that nPKCs (calcium independent)mediate activation-induced ROS generation. Despite the fact that thenPKC isoform PKCδ is involved in ROS generation in keratinocytes andmyeloid leukaemia cells (39, 43), we show here that PKCθ is essentialfor activation-induced ROS production in T cells. Moreover, it is knownthat PKCθ is crucial for T cell development and activation of thetranscription factors AP-1 and NF-κB (48, 59). These transcriptionfactors are major regulators of CD95L expression (26). In addition, AP-1as well as NF-κB are ROS sensitive (17). Thus, these data are in linewith the important role of PKCθ in oxidative signalling addressed by us.

PKCs are known to activate NOX2. Recently, it has been shown that humanand murine T cells express NOX2. T cells from mice deficient in thesesubunits displayed a reduced ROS production upon TCR stimulation (30).Here we demonstrate that NADPH oxidases participate inactivation-induced ROS generation in human T cells. Comparable to murineT cells, the oxidative signal is only partially NADPH oxidase dependent(FIG. 9H). Therefore, we focused on the identification of an additionalsource of ROS. Mitochondria are the most prominent intracellular sourceof ROS production (53). It has been reported that PKCs are translocatedinto/to mitochondria after PMA treatment (39, 43). Here we show thatupon activation PKCθ is translocated to the mitochondria and/orassociated membrane structures. In addition, mitochondria translocate tothe plasma membrane and the immunological synapse upon T cell activation(49). To analyse the role of mitochondria in activation-induced ROSgeneration in more detail we used cells transiently depleted of mtDNA,pseudo-ρ⁰ cells (12, 36). These cells not only reveal a diminishedactivation-induced oxidative signal but also a reduction of AICD.Therefore, we demonstrate for the first time that expression ofmitochondrial encoded proteins is a prerequisite for induction of AICD.

The ETC components are oligomeric complexes consisting of both nuclearand mtDNA-encoded subunits. The primary sites for mitochondrial ROSproduction by the ETC are complexes I and III (45). It has been shownthat rotenone, a commonly used inhibitor of complex I, interferes withCD8⁺ T cell function (70) and activation-induced CD95L expression (7).Nevertheless, rotenone inhibits, in addition, spindle microtubuleformation and tubulin assembly leading to cell cycle arrest, disassemblyof the Golgi apparatus, disturbance of the cytoskeleton andtubulin-dependent cell signalling events (4, 5, 8, 16, 32, 44, 52).Therefore, it is likely that rotenone interferes with formation of theimmunological synapse and movement of mitochondria.

However, here we prove the role of complex I in activation induced ROSproduction, CD95L expression, and AICD via downmodulation of NDUFAF1expression (FIG. 9H). Moreover, we exclude the participation of theother complexes of the ETC in activation-induced ROS production by theuse of different inhibitors. Thus, we show here that it is indeedcomplex I which generates ROS and is therefore responsible for inductionof CD95L expression and AICD. It is under discussion whether it is O₂ ⁻(15) or H₂O₂ (25) which acts as a second messenger in CD95L expressionand AICD. However, complex I is known to generate O₂ ⁻ into themitochondrial matrix (65). In aqueous solutions O₂ ⁻ has a half lifetime of less than 1 μs and is converted into H₂O₂ rapidly (53). MnSOD,an enzyme located the mitochondrial matrix, facilitates the conversionof O₂ ⁻ into H₂O₂ further. Here we show that MnSOD expression andactivity is enhanced upon TCR stimulation. Thus, O₂ ⁻ generated bycomplex I is converted into H₂O₂ that can cross the mitochondrialmembrane and acts then as a second messenger in the cytosol. Blockage ofcomplex I via inhibitors and siRNA-mediated downmodulation of NDUFAF1expression leads to a nearly complete block of ROS generation.Therefore, complex I activity is crucial for subsequent NADPH oxidasedependent ROS production (FIG. 9H). Recently, a similar connectionbetween mitochondrial ROS generation and activation of NOX1 has beendescribed in 293T cells (38). Thus, we demonstrate that ROS produced bymitochondria, despite being known as a damaging by-product ofrespiration, can also be released in a controlled process and serve as asecond messenger.

Next, we searched for potential tools to manipulate generation of ROS atcomplex I and verify weather our findings have possible applications intreatment of CD95/CD95L dependent diseases. Therefore, we analysed theeffect of metformin, an anti-diabetic drug (2, 3, 58) and mild inhibitorof complex I (6, 18) on AICD. Here we demonstrate that metformininhibits activation-induced ROS production and thereby CD95L expressionand AICD. It has been shown in vitro that metformin inhibits reversedelectron flux towards complex I (6). Therefore, we assume thatactivation-induced ROS production is coupled to reversed electrontransport. Importantly, metformin is a non-toxic complex I inhibitor andtherefore, a potential tool to investigate diseases displaying defectsin mitochondrial function combined with deregulation of CD95Lexpression.

AICD guards against development of autoimmunity. Thus, the pathology ofa recently reported case of fatal neonatal-onset mitochondrialrespiratory chain disease with manifestation of T cell immunodeficiency(51) could possibly be explained by our findings. Furthermore, multiplesclerosis (MS) is generally considered to be an inflammatory diseasewith a substantial autoimmune contribution. On the one hand, it wasshown in a genetic screening that about 20% of MS patients revealedmutations in their mtDNA (34). It was also stated that mitochondrialcomplex I gene variants are associated with MS (67). On the other hand,many patients suffering from LHON (Leber's hereditary optic neuropathy)disease caused by mutations in the mitochondria-encoded subunits ofcomplex I display symptoms of MS (33). The MS pathology in patients withmutations in genes of complex I is not understood so far, therefore, ourdata warrant to investigate whether CD95L expression may play a role inits development. The same applies to the T cell specificimmunodeficiency disorder associated with purine nucleosidephosphorylase deficiency, which is a result of inhibition of mtDNArepair due to the accumulation of dGTP in mitochondria (1). Since CD95Lplays an important role in T cell development, mitochondrial damage maybe responsible for impaired thymocyte differentiation in this disease.In addition, several T cell dependent diseases are associated withenhanced ROS levels e.g. lupus erythematosus (47), rheumatoid arthritis(22), and AIDS (25), which influence T cell activation, death andhomeostasis. Thus, the presented findings might have furtherimplications for the development of non-toxic inhibitors of complex I totreat diseases where deregulation of CD95L expression or T-cellactivation plays a vital role.

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The invention claimed is:
 1. A method of treating a patient having aT-cell mediated inflammatory disease, the method comprisingadministering to said patient a therapeutically effective amount of acompound which inhibits complex I-mediated ROS production.
 2. The methodaccording to claim 1, wherein the compound is capable of inhibitingreversed electron flux.
 3. The method according to claim 2, wherein thecompound belongs to the class of biguanides.
 4. The method of claim 3,wherein the biguanide is metformin or a pharmaceutically acceptable saltthereof.
 5. The method according to claim 1, wherein the compound iscapable of destabilizing complex I.
 6. The method according to claim 5,wherein the compound comprises a siRNA molecule wherein the siRNAmolecule interferes with the expression of NDUFAF1.
 7. The methodaccording to claim 1, wherein the T-cell mediated inflammatory diseaseis selected from the group consisting of graft-versus-host disease,lupus erythematosis, sepsis, asthma, psoriasis, atopical dermatitis, andmultiple sclerosis.
 8. A medicament against a T-cell mediatedinflammatory disease, the medicament containing a therapeuticallyeffective amount of a combination of compounds which inhibit complexI-mediated ROS production.
 9. The medicament of claim 8, wherein thecompounds which inhibit complex I-mediated ROS production are metforminor a pharmaceutically acceptable salt thereof and a compound which iscapable of destabilizing complex I.